Abstract Blood vessels are important for the distribution of nutrients and oxygen to all parts of our bodies. A hierarchical organization of differently sized blood vessels is key for their functionality. Several diseases can affect blood vessel topologies and diameters, leading to vascular malformations in humans. Previous reports suggested that increases in blood vessel diameters can be caused by increases in endothelial cell numbers, the cells of the blood vessels? inner lining. We established an imaging approach in zebrafish embryos that allows us to precisely analyze endothelial cell numbers in addition to their shapes and sizes during embryonic development. Surprisingly, we found that initially the shapes and sizes of endothelial cells were more critical for blood vessel diameter control than their numbers and that endothelial cell numbers increased only at later stages of vascular malformation establishment. Importantly, we also found that endothelial shapes and sizes were affected in mutants of several different genes causing vascular malformations. However, to date it is not known how these genes affect cell shapes and sizes and how this would impact blood vessel diameters. The aims in this proposal address this question by analyzing the cellular and molecular components influencing cell shapes and sizes. In aim 1 we will investigate how the cytoskeleton and its contractile properties affect cellular dimensions and how this might feedback on blood vessel diameters. We will also investigate whether changing the cytoskeleton and hence endothelial cell contractility can rescue vascular malformations. In aim 2 we plan to interrogate the influence of endothelial cell polarity on blood vessel diameters. Endothelial cells have an apical, facing the blood vessel lumen, and a basolateral membrane domain. At present, we do not know how mutations causing vascular malformations change apical-basal polarity and how these changes would affect blood vessel diameters. We will test for these possibilities by examining apical and basolateral polarity in different zebrafish mutants that develop vascular malformations. We will also investigate how changing apical-basolateral polarity will affect endothelial cell shapes and sizes in these mutants. Ultimately, we aim to reverse vascular malformations through normalizing apical-basolateral polarity and endothelial cell contractility and thereby endothelial cell shapes prior to increases in endothelial cell numbers.